It has been established that the phenotypic expression and survival of differentiating neural cells and the survival and metabolism of established neural cells is directed by a variety of extracellular signals. Neighboring cells and processes surrounding neural cells, play an important role in the regulation of cell differentiation, metabolic function and survival, largely through their release of growth and other regulatory factors.
Many neurological disorders, including Parkinson's disease, are the result of the degeneration of specific groups of neural cells. In the case of Parkinson's disease, the degeneration of a group of dopamine-containing cells, which connect the ventral tegmentum and substantia nigra (located in the ventral portion of the mesencephalon, or midbrain) with the striatum, have been implicated in the etiology of the condition.
In order to understand the factors which result in, or could prevent, the degeneration of these dopaminergic pathways, tissue obtained from the mesencephalon region has been extensively studied. Embryonic dopamine-containing neurons, derived from mesencephalic tissue, are difficult to culture as the dopaminergic neurons do not survive well in culture. However, these cultures show an enhanced survival and/or a modified biochemical activity when cultured with a conditioned medium or when treated with growth factors. Embryonic tissue from the mesencephalon has been grown in conditioned culture media (CM) derived from the rat B49 glial cell line, the R 33 neural retina glial cell line and the J S Schwannoma cell line [Engele, J., et al., J. Neurosci. Res., 30: 359-371, (1991)]. In all three cases, CM significantly increased the survival of the cultured dopaminergic neurons. The enhanced survival of the dopaminergic neurons was not due to the proliferation of dopaminergic cells but was attributed to the effects of the CM on existing glial cells derived from the mesencephalic culture and the resultant interactions between the glial cells and the dopaminergic neurons, rather than to a direct effect on the dopaminergic neurons.
Culturing embryonic mesencephalic tissue in CM prepared from mesencephalic astrocytes, or co-culturing the tissue on a layer of mesencephalon-derived astrocytes, rescues dopaminergic neurons from death induced by serum deprivation. Astrocytes or the CM prepared from astrocytes from the striatum and cerebral cortex had significantly weaker protective effects [Takeshima et al. J. Neurosci., 14(8): 4769-4779, (1994)]. In one report, CM derived from cortical astrocytes had no effect on dopaminergic cell survival or proliferation but did alter the biochemistry of this population of cells, resulting in a small increase in their uptake of dopamine [Gaul and Lubbert, Proc. R. Soc. Lond. B, 249: 57-63, (1992)].
Growth factors, many of which are present in CM from neural tissue, are believed to be responsible for regulating dopaminergic neuron survival and metabolism, either directly, or through their effects on adjacent cells. Growth factors reported to have a direct effect on dopaminergic neuron survival include interleukin-6 (IL-6) [Hama et al., Neurosci., 40(2): 445-452 (1991)], brain-derived neurotrophic factor (BDNF) [Hyman et al., Nature, 350: 230-232, (1991)], basic fibroblast growth factor (FGF-2, formally referred to as bFGF) [Dal Toso et al. J. Neurosci., 8(3): 733-745 (1988); Ferrari et al. Dev. Biol. 133: 140-147 (1989); and glial cell line-derived neurotrophic factor (GDNF), secreted by the rat B49 cell line [Lin, L. G., et al, Science, 260: 1130-1132 (1993)], all of which specifically enhance the survival of dopaminergic neurons in dissociated rat or mouse embryo mesencephalon cultures without increasing neuron or glial cell numbers. GDNF dramatically increases the morphological differentiation of dopaminergic neurons, resulting in more extensive neurite outgrowth and increased cell body size. Some growth factors, such as nerve growth factor (NGF) (Hatanaka and Tsuki (1986), Dev. Brains Res. 30: 47-56), platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) (Engele & Bohn (1991), Neurosci. 11(10): 3070-3078); Mayer, E. (1993) Dev. Brain Res. 72: 253-258) and nerve growth factor (NGF) (Engele & Bohn, ibid) are reported to support dopaminergic cell survival in embryonic mesencephalic tissue through a glial cell-mediated mechanism.
In vivo studies indicate that the damage caused by mechanical or chemical lesions in the dopaminergic pathways between the mesencephalon and the striatum can be significantly reduced by treatment with epidermal growth factor (EGF) [Pezzoli et al, Movement Disorders 6(4) 281-287, (1991)] and BDNF (Hyman et al., supra). In vitro treatment with cyclic AMP, but not FGF-2 or NGF, increased the survival of cultured mesencephalic dopaminergic neurons in response to chemically induced degeneration produced by 1-methyl-4-phenylpyridinium (MPP.sup.+) [Hyman et al., supra; Hartikka et al., J. Neurosci. Res., 32: 190-201, (1992)]. Although the use of a FGF-2-stimulated astrocyte CM enhances dopamine uptake, the use of this CM did not have a protective effect when the neurons were chemically lesioned using MPP.sup.+ (Gaul and Lubbert, supra).
Many of the cells obtained from embryonic tissue which normally gives rise to dopaminergic neurons (i.e. normally dopaminergic tissue), such as the mesencephalon and olfactory bulb, will eventually differentiate into dopaminergic neurons under primary culture conditions. However, an increased number of cells, in tissue obtained from normally dopaminergic areas of the brain, can be induced to differentiate into dopaminergic cells by co-culturing with feeder cell layers derived from neural tissue. The dopaminergic neurons of the olfactory bulb show a five-fold increase in number when embryonic olfactory bulb neurons are co-cultured with olfactory epithelial neurons. It is believed that a soluble factor, calcitonin gene-related peptide (CGRP), which is present in the epithelial cells, is responsible for the induction of additional dopaminergic neurons in the olfactory bulb [Denis-Donini, Nature, 339: 701-703, (1989)]. Co-culturing rat embryonic neostriatal and substantia nigral tissue for one to three weeks on glial cell feeder layers obtained from the substantia nigra region induces the expression of dopaminergic cells as indicated by tyrosine hydroxylase immunoreactivity (TH+) in the tissue cultured from both areas. However, when the same tissues were co-cultured with glial cells from the neostriatum, dopaminergic cells were observed only in the substantia nigra tissue but not in the neostriatal tissue [Beyer et al., Neurosci. Lett., 128: 1-3, (1991)]. The mechanism underlying the appearance of TH-IR in neostriatal tissue (an area which does not contain dopaminergic cells in the adult) was not determined. However, it could have been due to the induction of TH+ cells in the substantia nigral cell feeder layer in response to the presence of the striatal tissue; to the induction of dopaminergic properties in the striatal cells; or to promotion of the survival of dopaminergic cells in striatal tissue, such cells having been reported to occur transiently during development in the striatum (Tashiro et al. (1989), Neurosci. Lett. 97: 6-10) and cortex (Satoh and Suzuki (1990), Dev. Brain Res. 53: 1-5). Small numbers of TH+ cells (140 TH+ cells/cm.sup.2) have been induced in tissue from embryonic rat cortex using a combination of BDNF and dopamine, in a culture medium which contained 10% fetal calf serum. Fewer TH+ cells were seen when BDNF, or dopamine were used alone (Zhou et al. (1994), Dev. Brain Res. 81: 318-324). A few cells from embryonic mouse striatal tissue can be induced to express TH+ when incubated with FGF-1 and an enhanced result can be obtained using a combination of FGF-1 and an unidentified &gt;10 kD fraction obtained from muscle tissue (Du et al. (1994) J. Neurosci. 14(12): 7688-7694).
The degeneration of the substantia nigra dopaminergic neurons which characterizes Parkinson's Disease is normally treated using pharmacological interventions to augment the declining natural dopamine supply to the striatum. However, there are problems associated with drug treatment such as the development of tolerance to the medication and possible side effects. Neuronal grafts, using embryonic substantia nigral tissue have shown some potential for relieving experimentally induced Parkinsonism in rodents and primates and in some human Parkinsonian patients. However, graft survival is poor and only limited quantities of embryonic dopaminergic tissue are available. On average, 4-10 fresh, human embryos are required to obtain sufficient numbers of dopaminergic neurons for a single human transplant (Widner et al., N Engl J Med 327: 1556-1563 (1992)). Preferred treatment would involve prevention of, or a reduction in the amount of the degeneration which occurs. Once damage has occurred, it would be preferable to replace the lost cells by implanting new dopaminergic neurons using cells derived from neural cells which have been proliferated in culture preferably from a non-tumor cell line, or from cells that have not been intentionally immortalized in order to induce proliferation and, most preferably, would be derived from a patient's own neural tissue. Alternatively, a less invasive treatment would involve the in vivo manipulation of the patient's own population of neural cells in order to replace the function of the damaged dopaminergic neurons.
The prior art suggests that cultures of dopaminergic cells can be obtained through the use of glial feeder layers, or the application of certain growth factors or conditioned media to mesencephalic tissue and other dopaminergic tissues. These treatments can induce differentiation, increase the survival, or alter the metabolism of cells from normally dopaminergic tissue that has been cultured in vitro. However, culture methods for inducing cells from other, non-dopaminergic brain regions to differentiate into dopaminergic cells are limited. While it has been demonstrated that a feeder layer of cells from regions such as the substantia nigra and olfactory bulb (areas which normally contain a relatively high population of dopaminergic cells) can be used to induce the appearance of dopaminergic cells in certain embryonic central nervous system (CNS) tissues, there is no evidence that cells from non-dopaminergic neural tissue could be used as feeder layers in tissue culture designed to induce the appearance of dopamine in tissues, such as the striatum, which do not normally contain dopamine.
For some purposes, especially transplantation and certain drug testing procedures, it would be advantageous to use completely defined culture conditions to induce the differentiation of dopaminergic cells. It would be particularly advantageous if the cells were obtained from both dopaminergic and normally non-dopaminergic neural tissue sources, thus maximizing the number of dopaminergic cells that can be generated from a single embryo.
There exists a need in the art for a reliable method of inducing neural cells, derived from all brain regions, from tissue obtained from animals of all ages, to differentiate into dopaminergic cells in the presence or absence of a feeder layer substrate. In particular, it would be advantageous to induce the expression of dopamine in cells derived from regions which do not normally contain dopaminergic cell bodies, such as the striatum, but which require dopamine for normal functioning.
Recently, it has been demonstrated that multipotent neural stem cells, obtained from embryonic, juvenile, and adult tissue, can be proliferated in vitro to generate large numbers of neural stem cell progeny, which, under appropriate conditions, can differentiate into neurons and glia (PCT applications No. WO 93/01275, WO 94/16718, WO 94/10292, and WO 94/09119). It would be advantageous to generate dopaminergic cells from the proliferated progeny of multipotent neural stem cells, derived from any area of the CNS.
Accordingly, it is an object of this invention to provide a method for inducing large numbers of neural cells obtained from normally non-dopaminergic tissue to differentiate, in vitro, into dopaminergic cells in order to provide a reliable source of dopaminergic cells for various applications such as transplantation into patients with dopamine deficiencies and for drug screening procedures.
It is a further object of the invention to provide a method of inducing the undifferentiated, proliferated progeny of multipotent neural stem cells, derived from any area of the CNS known to contain such cells, to differentiate into dopaminergic cells, in order to provide unlimited quantities of dopaminergic cells for transplantation, drug screening and other purposes.
Additionally, it is an object of the invention to provide tissue culture methods that use completely defined culture conditions, and thus do not require the presence of a feeder layer of cells, conditioned medium, or serum, to increase the number of dopaminergic cells obtained from a single embryonic brain. Such cells would have use in specific applications such as transplantation into patients with dopamine deficiencies and for certain drug screening procedures.
These and other objects and features of the invention will be apparent to those skilled in the art from the following detailed description and the appended claims.
None of the foregoing references is believed to disclose the present invention as claimed and is not presumed to be prior art. The references are offered for the purpose of background information.